Crystalline polymorphic form of (S,S,S)-N-(1-[2-carboxy-3 (N2-mesyllyslamino) propyl]-1- cyclopentylcarbonyl) tyrosine

ABSTRACT

The present invention relates to a crystalline, α-polymorphic form of a compound of formula (I) and to processes for the preparation of, to intermediates used in the preparation of, to compositions containing and to uses of, the α-polymorphic form.

This application was filed under 35 U.S.C. §371 based on PCT/EP94/03750,which was filed on Nov. 9, 1994 which claims priority from U.K.application serial no. 9324931.6 which was filed on Dec. 4, 1993 and isnow abandoned.

The present invention relates to a crystalline, polymorphic form of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinewhich has the formula:

hereafter referred to as the “α-form” of a compound of the formula (I).

More particularly, the invention relates to the α-form of a compound ofthe formula (I) and to processes for the preparation of, tointermediates used in the preparation of, to compositions containing andto uses of, the α-form.

An amorphous form (hereafter referred to as the “β-form”) of a compoundof the formula (I) has been disclosed in European Patent Publication No.EP-A-0358398 as Example 181. The compound is a potent inhibitor of thezinc dependent neutral endopeptidase E.C.3.4.24.11 and is therefore ableto potentiate the biological effects of atrial natriuretic factor. It istherefore a natriuretic, antihypertensive and diuretic agent that isuseful for the treatment of various cardiovascular disorders. Thecompound is also a potent inhibitor of angiotensin converting enzyme, afurther enzyme that is involved in the control of blood pressure. Thecompound therefore has a dual pharmacological action through beingcapable of inhibiting two key enzymes involved in the control of bloodpressure. It is therefore likely to be useful in the treatment ofvarious forms of hypertension and associated cardiovascular disorderssuch as congestive heart failure and glaucoma.

The β-form can be obtained by methods such as freeze drying of asolution of the compound of the formula (I), by rapid evaporation of thesolvent from such a solution or by precipitation from such a solution byaddition of a poor solvent. The β-form does not melt sharply butnormally “softens” at about 160° C.

The β-form has, however, been found to have certain properties which donot make it particularly suitable for pharmaceutical formulation. Inparticular it is hygroscopic in nature, it has a low bulk density andpoor flow properties. Processing experiments carried out using theβ-form have revealed problems in manufacturing tablets from compositionscontaining this form.

The problem addressed by the present invention is the provision of aform of the compound of the formula (I) which can be efficientlyprocessed to provide a stable and effective formulation of the drug.

This problem has been solved by the surprising finding that an α-form ofa compound of the formula (I) can be prepared which is non-hygroscopic,crystalline and, when compared to the β-form, which has a higher bulkdensity and better flow properties. The α-form is particularly suitablefor use in pharmaceutical formulation of the drug.

The present invention therefore provides a crystalline, polymorphicα-form of a compound of the formula (I) which has an infra-red spectrumas a mull in nujol which shows absorption bands at ν=3407, 3386, 3223,3153, 1699, 1652, 1626, 1594, 1516, 1457 (nujol), 1377 (nujol), 1344,1334, 1317, 1267, 1241, 1228, 1210, 1164, 1151, 1137, 1118, 1109, 1093,1074, 1045, 1019, 1003, 981, 965, 911, 897, 862, 818, 800, 778, 762, 721and 655 cm⁻¹.

The α-form is further characterised by its powder X-ray diffractionpattern obtained using copper radiation filtered with a graphitemonochromator (λ=0.15405 nm) which shows main peaks at 7.5, 8.9, 9.9,11.6, 15.6, 17.2, 17.5, 18.0, 20.2, 22.1 and 23.3 degrees 2θ.

The α-form is yet further characterised by differential scanningcalorimetry in which it shows a sharp endotherm in the range 248-259° C.and decomposes at above 260° C. when subjected to a scanning rate of 20°C. per minute.

The α-form typically melts sharply in the range 242-252° C., althoughlower melting point ranges have been recorded.

Other forms (hereafter referred to as the “γ-” and “δ-forms”) of acompound of the formula (I) have also been obtained which also form partof the present invention since they can be used as intermediates in thepreparation of the α-form.

The invention thus further provides a polymorphic γ-form of a compoundof the formula (I) which has an infra-red spectrum as a mull in nujolwhich shows absorption bands at ν=3377, 3240, 1665, 1639, 1594, 1527,1518, 1494, 1457 (nujol), 1443, 1377 (nujol), 1344, 1321, 1304, 1254,1195, 1178, 1162, 1143, 1111, 1098, 1046, 1031, 1012, 972, 962, 945,932, 907, 879, 849, 815, 806, 780, 753, 729 and 658 cm⁻¹.

The γ-form is further characterised by its powder X-ray diffractionpattern obtained using copper radiation filtered with a graphitemonochromator (λ=0.15405 nm) which shows main peaks at 9.0, 9.6, 10.6,11.6, 12.7, 13.3, 14.6, 16.2, 17.9, 18.8, 20.2 and 21.8 degrees 2θ.

The γ-form is yet further characterised by differential scanningcalorimetry in which it shows a sharp endotherm in the range 176-186°C., an exotherm at about 207° C. and a weak endotherm at about 213° C.and decomposes at above 250° C. when subjected to a scanning rate of 20°C. per minute.

The γ-form typically melts sharply in the range 170-185° C.

The invention thus also provides a hydrated 6-form of a compound of theformula (I) which has a water content of from 1 to 7%, preferably offrom 2 to 4%, by weight, as determined by Karl Fischer analysis, andwhich has an infra-red spectrum as a mull in nujol which showsabsorption bands at ν=3667, 3425, 3380, 3287, 3137, 3098, 1709, 1673,1637, 1619, 1596, 1568, 1556, 1516, 1458 (nujol), 1448, 1419, 1390, 1378(nujol), 1356, 1338, 1300, 1270, 1249, 1229, 1198, 1174, 1141, 1108,1091, 1075, 1064, 1045, 1033, 1019, 1001, 985, 962, 941, 909, 889, 877,841, 822, 807, 763, 744, 732, 721 and 655 cm⁻¹.

The δ-form is further characterised by its powder X-ray diffractionpattern obtained using copper radiation filtered with a graphitemonochromator (λ=0.15405 nm) which shows main peaks at 10.5, 10.8, 12.3,14.5, 17.2, 17.6, 17.9, 18.9, 20.4, 21.5, 22.4, 23.0, 23.1, 24.7, 27.1,27.8 and 28.9 degrees 2θ.

The δ-form is yet further characterised by differential scanningcalorimetry in which it shows sharp endotherms at about 162° C. and atabout 166-168° C. and decomposes at above 200° C. when subjected to ascanning rate of 20° C. per minute.

The δ-form typically melts sharply in the range 165-175° C.

Although the γ- and δ-forms of a compound of the formula (I) display thesame pharmacological activities as the α- and β-forms, they are not assuitable as the α-form for the purpose of pharmaceutical formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B are IR spectra for certainpolymorphs of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine.

FIGS. 5-8 are PXRD patterns of certain polymorphs of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine.

FIGS. 9-12 are DSC thermographs for certain polymorphs of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine.

FIGS. 13-15 describe processing data for certain polymorphs of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine.

FIGS. 16-17 describe hygrscopicity data for certain polymorphs of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine.

The α-form of a compound of the formula (I) can be prepared by thefollowing methods:

1) The α-form can be prepared by catalytic hydrogenation of an aqueoussolution of a sodium, potassium, ammonium or (C₁-C₄ alkyl)ammonium saltof a compound of the formula:

 using a suitable catalyst for the removal of the benzyloxycarbonylprotecting group, e.g. palladium-on-carbon, followed by acidification ofthe base salt of the compound of the formula (I) obtained to from pH 3to 5, preferably to about pH4, and preferably at from 35 to 45° C., toprovide the α-form. Preferably a disodium salt of a compound of theformula (II) is used. Further suitable catalysts for the removal of thebenzyloxycarbonyl protecting group are well known to the skilled person,e.g. see T. W. Greene and P. G. Wuts, “Protective Groups in OrganicSynthesis”, Second Edition, 1991, the teaching of which is incorporatedherein by reference.

In a typical procedure, a solution of a compound of the formula (II) ina suitable organic solvent, e.g. ethyl acetate, is shaken with anaqueous solution of sodium hydroxide to generate a disodium saltthereof. The aqueous solution containing the sodium salt is thenseparated and hydrogenated in the presence of a 5% palladium-on-carboncatalyst at about 414 kPa (60 psi) and room temperature to remove thebenzyloxycarbonyl protecting group. The catalyst is then removed byfiltration and the filtrate adjusted to about pH 4 using a suitableacid, e.g. aqueous hydrochloric acid. The α-form is precipitated fromsolution and can be collected by filtration.

A compound of the formula (II) can be prepared by the route set out inScheme 1.

 In a typical procedure, t-butyl acrylate (III) is reacted withparaformaldehyde in the presence of 3-quinuclidinol to provide t-butylhydroxymethylacrylate (IV). This is first treated with thionyl chloridein the presence of triethylamine and pyridine to provide thecorresponding chloromethylacrylate, which is then reacted with(S,S)-α-α′-dimethyldibenzylamine to provide an acrylate of the formula(V). This is converted to a compound of the formula (IX) by the methodset out in Tet. Lett., 1993, 34(8), 1323-6. A compound of the formula(IX) is then condensed with a lysine derivative of the formula (X) by asimilar procedure to that described in EP-A-0358398 for the preparationof a compound of the formula (XI). A compound of the formula (XI) isthen converted to a compound of the formula (II) using a solution oftrifluoroacetic acid and anisole in dichloromethane.

2) The α-form can be prepared from the δ-form by stirring a solution ofthe δ-form in water or in an aqueous solution of a suitable organicsolvent, e.g. a C₁-C₄ alkanol such as methanol or isopropanol, or aC₃-C₆ alkanone such as acetone.

In a typical procedure the δ-form is dissolved in a 1:5 water/methanolor a 1:10 water/acetone mixture and the solution stirred for severaldays at room temperature. The α-form precipitates from the solution andcan be collected by filtration.

3) The α-form can be prepared from the γ-form by stirring a solution ofthe γ-form in water or in an aqueous solution of a suitable organicsolvent, e.g. a C₁-C₄ alkanol such as methanol or isopropanol, or aC₃-C₆ alkanone such as acetone.

In a typical procedure the γ-form is dissolved in a 1:1 water/methanolmixture and the solution stirred for about 17 hours at room temperature.The α-form precipitates from the solution and can be collected byfiltration.

4) The α-form can be prepared from the β-form by a similar procedure tothat set out in Method (3) above.

5) The α-form can be prepared by deprotection, preferably under acidicconditions, of a compound of the formula:

 wherein P¹, P², P³ and P⁴ are all suitable protecting groups that arecapable of removal, preferably under acidic conditions, to provide,following adjustment of the pH to from 3 to 5, preferably about 4, inthe work-up, the α-form.

Suitable protecting groups for this purpose together with conditions fortheir removal will be well known to the skilled person, e.g. see T. W.Greene, and P. G. Wuts, “Protective Groups in Organic Synthesis”, SecondEdition, Wiley-Interscience. P¹ is preferably formyl orbenzyloxycarbonyl. P², P³ and P⁴ are preferably each t-butyl.

In a typical procedure where P¹ is formyl or benzyloxycarbonyl and P²,P³ and P⁴ are each t-butyl, a solution of a compound of the formula(XII) in a suitable solvent, e.g. 1,4-dioxane or ethyl acetate, istreated with a suitable acid, e.g. hydrogen chloride, to remove to theprotecting groups and adjustment of the pH to about 4 in the work-upprovided the α-form.

The intermediates of the formula (XII) may be prepared by conventionaltechniques. The compound of the formula (XlI) where P¹ isbenzyloxycarbonyl and P², P³ and P⁴ are each t-butyl corresponds to thecompound of the formula (XI) in Scheme 1, the synthesis of which isfurther described in Method (1). The compound of the formula (XII) whereP¹ is formyl and P², P³ and P⁴ are each t-butyl may be prepared by firstremoving the benzyloxycarbonyl group from the compound of the formula(XI) by hydrogenolysis using a suitable catalyst, e.g.palladium-on-carbon, followed by formylation of the amine obtained, e.g.using formic acetic anhydride.

6) The α-form can be prepared by deprotection, preferably under acidicconditions, of a compound of the formula:

 wherein P⁵ is a suitable protecting group that is capable of removal,preferably under acidic conditions, to provide, following adjustment ofthe pH to from 3 to 5, preferably about 4, in the work-up, the α-form.Suitable protecting groups for this purpose together with conditions fortheir removal will be well known to the skilled person, e.g. see T. W.Greene and P. G. Wuts, “Protective Groups in Organic Synthesis”, SecondEdition, Wiley-Interscience. P⁵ is preferably formyl and furtherexamples of P⁵ are benzyloxycarbonyl and tert-butyloxycarbonyl.

In a typical procedure where P⁵ is formyl, a solution of a compound ofthe formula (XIII) in a suitable solvent, e.g. 1,4-dioxane, is treatedwith an aqueous solution of a suitable acid, e.g. hydrochloric acid, toremove the protecting group and adjustment of the pH to about 4 inwork-up provided the α-form.

The intermediates of the formula (XIII) may be prepared by conventionaltechniques, such as by selective deprotection of a compound of theformula (XII) to remove the P², P³ and P⁴ protecting groups alone. Forexample, where P¹ is formyl and P², P³ and P⁴ are each t-butyl, thet-butyl protecting groups may be selectively removed by treatment of acompound of the formula (XII) with trifluoroacetic acid in a suitablesolvent, e.g. dichloromethane.

The β-, γ- and δ-forms that are used as intermediates in preparing theα-form can be prepared as follows:

(i) The β-form can be prepared by catalytic hydrogenation of a solutionof a compound of the formula (II) in a suitable solvent and in thepresence of a suitable catalyst for the removal of the protecting group,e.g. palladium-on-carbon.

In a typical procedure, a solution of a compound of the formula (II) inaqueous ethanol is hydrogenated at about 414 kPa (60 psi) and roomtemperature in the presence of a palladium-on-carbon catalyst. Thecatalyst is then removed by filtration and the filtrate is eitherconcentrated under reduced pressure to provide a foam that is stirredwith a C₃-C₆ alkanone, e.g. acetone, or freeze dried, to provide theβ-form that can be collected by filtration. This preparation has also,if the C₃-C₆ alkanone treatment is used, occasionally provided theα-form.

(ii) The δ-form can be prepared by catalytic hydrogenation of a solutionof a compound of the formula (II) in a mixture of a suitable waterimmiscible organic solvent, e.g. ethyl acetate, and water and in thepresence of a suitable catalyst for the removal of the protecting group,e.g. palladium-on-carbon, followed by removal of the catalyst,separation of the aqueous layer and precipitation of the product fromthe aqueous layer using a C₁-C₄ alkanol, e.g. methanol.

In a typical procedure, water is added to a solution of a compound ofthe formula (II) in ethyl acetate and the mixture is hydrogenated atabout 414 kPa (60 psi) and room temperature in the presence of apalladium-on-carbon catalyst. The catalyst is then removed byfiltration, the aqueous phase separated from the filtrate, concentratedunder reduced pressure to a low volume and poured into methanol. The6-form slowly precipitates from the solution and can be collected byfiltration.

This preparation has also occasionally provided the α-form.

(iii) The β-form can be prepared by first freezing an aqueous solutionof the β-form and then freeze drying the resulting solid mass.

(iv) The γ-form can be prepared by stirring the δ-form with n-propanolor acetonitrile.

In a typical procedure the mixture is stirred for about 24 hours at roomtemperature and the γ-form is collected by filtration.

(v) The γ-form can be prepared by stirring a slurry of the β-form inacetonitrile or n-propanol, typically for about 5 days at roomtemperature. The γ-form is collected by filtration.

(vi) The γ-form can be prepared by treating an aqueous solution of theδ-form with a C₃-C₆ alkanone, e.g. acetone.

In a typical procedure an aqueous solution of the δ-form is poured intoa vigorously stirred volumetric excess of acetone at room temperature.The γ-form precipitates from solution and can be collected byfiltration.

This preparation has also occasionally provided the α-form.

(vii) The β-form can be prepared by freeze drying a concentrated,aqueous solution of the α-form.

In a typical procedure, a concentrated solution of the α-form in hotwater is prepared, the solution filtered to remove any insolublematerial, then cooled, frozen and finally freeze dried to provide theβ-form.

As previously mentioned, the α-form is a potent inhibitor of the neutralendopeptidase (E.C.3.4.24.11). This enzyme is involved in the breakdownof a number of peptide hormones and peptide autocoid substancesincluding, in particular, the breakdown of atrial natriuretic factor(ANF). Thus the α-form, by preventing the degradation of ANF by neutralendopeptidase E.C.3.4.24.11, can potentiate the biological effects ofANF and is therefore a diuretic, natriuretic and antihypertensive agentof utility in the treatment of a number of disorders includinghypertension, heart failure, angina, renal insufficiency, chronic renalfailure, premenstrual syndrome, cyclical oedema, Menieres disease,hyperaldosteroneism (primary and secondary) and hypercalciuria. Inaddition, because of its ability to potentiate the effects of ANF, theα-form is useful in the treatment of glaucoma. Further, as a result ofits ability to inhibit the neutral endopeptidase E.C.3.4.24.11, theα-form may be useful in treating asthma, inflammation, pain, epilepsy,affective disorders, dementia, geriatric confusion, obesity,gastrointestinal disorders (especially diarrhoea and irritable bowelsyndrome) and hyperreninaemia and in the modulation of gastric acidsecretion.

The activity against neutral endopeptidase E.C.3.4.24.11 can be assessedusing a procedure based on the assay described by Barclay, P. L., et al,Biochem. Biophys. Res. Comm., 1989, 164, 58-65. The method involvesdetermining the concentration of compound required to reduce by 50% therate of release of radiolabelled hippuric acid fromhippuryl-L-phenylalanyl-L-arginine by a neutral endopeptidasepreparation from rat kidney.

As previously mentioned, the α-form is also an inhibitor of angiotensinconverting enzyme (ACE). As such it is useful in treating a variety ofconditions for which ACE inhibitors are known to be useful includinghypotension, congestive heart failure, limitation of ischaemic damage tothe myocardium, protection of the kidney against hyperfiltration damage,prevention or reversal of left ventricular hypertrophy, memoryenhancement, control of cognitive function, dementia and preventingreocclusion following coronary angioplasty or coronary artery bypasssurgery. Its activity against this enzyme can be assessed using aprocedure which is based on a modification of the assay described byRohrbach, M. S., Anal. Biochem., 1978, 84, 272. The method involvesdetermining the concentration of compound required to reduce by 50% theextent of release of radiolabelled hippuric acid fromhippuryl-L-histidyl-L-leucine by angiotensin converting enzyme isolatedfrom the rat kidney.

Inhibitory activity can also be measured in vivo following intravenousinjection to anaesthetised rats using the methods described by I. L.Natoff et al, Journal of Pharmacological Methods, 1981, 5, 305 and by D.M. Gross et al, J. Pharmacol, Exp. Ther., 1981, 216, 552. The dose ofthe inhibitor that is required to reduce the pressor response producedby intravenous injection of angiotensin 1 (50 ng bolus) by 50% isdetermined.

The activity of the α-form as a diuretic agent can be determined bymeasuring its ability to increase urine output and sodium ion excretionin conscious AV-blocked dogs using the methods described by Alabaster,C. T., et al, Brit. J. Pharmacol., 1989, 98, 823P.

The antihypertensive activity of the α-form can be evaluated bymeasuring the fall in blood pressure following oral or intravenousadministration to salt depleted, diuretic primed, spontaneouslyhypertensive rats, salt depleted renally hypertensive dogs, ordesoxycorticosterone acetate (DOCA)/salt hypertensive rats.

For administration to an animal in the treatment of hypertension,congestive heart failure or renal insufficiency, oral dosages of theα-form will generally be in the range of 1-500 mg daily, and preferably5-200 mg daily for the treatment of human beings, for an average adultpatient. Thus for a typical adult human patient, individual tablets orcapsules contain from 1 to 200 mg of the compound in a suitablepharmaceutically acceptable diluent or carrier for administrationsingly, or in multiple doses, once or several times a day. Dosages forintravenous administration would typically be from 0.01 to 50 mg,preferably 0.1 to 10 mg, of compound per single dose as required. Inpractice the physician will determine the actual dosage which will bemost suitable for an individual patient and it will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case but there can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

For human use, the α-form can be administered alone, but will generallybe administered in admixture with a pharmaceutically acceptable diluentor carrier selected with regard to the intended route of administrationand standard pharmaceutical practice. For example, it may beadministered orally in the form of tablets containing such excipients asstarch or dibasic calcium phosphate, or in capsules or ovules eitheralone or in admixture with excipients, or in the form of an elixir or asuspension containing flavouring or colouring agents. It may be injectedparenterally, for example, intravenously, intramuscularly orsubcutaneously. For parenteral administration, it is best used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood.

The α-form may be co-administered with other agents that are useful forthe control of blood pressure, the treatment of cardiac conditions orrenal insufficiency. Thus, for example, it may be co-administered with acardiac stimulant, for example digitalis, an alpha-blocker, for exampledoxazosin, a beta-blocker, a calcium channel blocker, for exampleamlodipine, exogenous ANF, a potassium channel activator or with anotherdiuretic agent as shall be determined by the physician with regard tothe particular patient or disease state.

Therapeutic treatment by use of the α-form as disclosed herein can meancurative or prophylactic treatment of a particular disease.

The invention thus further provides:

(a) a pharmaceutical composition comprising the α-form, γ-form orhydrated δ-form of a compound of the formula (I) together with apharmaceutically acceptable diluent or carrier.

(b) the α-form, γ-form or hydrated δ-form of a compound of the formula(I), or a pharmaceutical composition thereof, for use as a medicament.

(c) the use of the α-form, γ-form or hydrated δ-form of a compound ofthe formula (I), or of a pharmaceutical composition thereof, for themanufacture of a medicament for treating a disease which is dependent onthe inhibition of angiotensin converting enzyme and/or zinc dependentneutral endopeptidase E.C.3.4.24.11.

(d) use as stated in (c) where the disease is a cardiovascular disordersuch as hypertension, congestive heart failure, renal insufficiency orglaucoma.

(e) a method of treatment of an animal, including a human being, totreat a disease which is dependent on the inhibition of angiotensinconverting enzyme and/or zinc dependent neutral endopeptidaseE.C.3.4.24.11, which comprises administering to said animal a saidenzyme and/or said endopeptidase inhibitory amount of the α-form, γ-formor hydrated δ-form of a compound of the formula (I) or a pharmaceuticalcomposition thereof.

(f) a method as stated in (e) where the disease is as stated in (d).

(g) a sodium, potassium, ammonium or (C₁-C₄ alkyl)ammonium salt of acompound of the formula (II).

(h) the γ-form of a compound of the formula (I).

(i) the hydrated δ-form of a compound of the formula (I).

(j) a compound of the formula (XII) with the proviso that P¹ is notbenzyloxycarbonyl when P², P³ and P⁴ are each t-butyl.

(k) a compound of the formula (XIII) with the proviso that P⁵ is notbenzyloxycarbonyl.

The preparation of the α-form is illustrated by the following Examples:

EXAMPLE 1(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

A solution of(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-carboxypropyl]-1-cyclopentylcarbonyl)tyrosinein ethyl acetate (1190 ml) (a portion of the solution obtained accordingto the method of Preparation 9 and taken to contain 219 g of thestarting material) was shaken with a solution of sodium hydroxide (23.1g) in water (503 ml). The aqueous phase was separated and hydrogenatedat 414 kPa (60 psi) and room temperature over a 5% palladium-on-carboncatalyst (20 g) for 5 hours. The catalyst was then filtered off and thefiltrate adjusted to pH 4 with 5N aqueous hydrochloric acid solution anda white solid precipitated. After granulating for 18 hours at roomtemperature, the solid product was filtered, washed with water and driedto give the title compound as a white solid (124.4 g), m.p. 248-250° C.Found: C,53.47; H,7.25; N,9.50. C₂₆H₄₀N₄O₉S requires: C,53.41; H,6.90;N,9.58%.

EXAMPLE 2(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

A solution of(S,S,S)-N-(1-[2-carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinehydrate (the δ-form, see Preparation 2) (3.0 g) in a 1:5 water/methanolmixture (18 ml) or a 1:10 water/acetone mixture (33 ml) was stirred for3 days at room temperature. The resulting solid was collected byfiltration and dried to give the title compound as a white solid, m.p.246-8° C. (from the aqueous methanol method), m.p. 242-3° C. (from theaqueous acetone method).

EXAMPLE 3(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,γ-form (see Preparations 4, 5, 7 and 8) (0.5 g) was dissolved in water(4 ml) and methanol (4 ml) was added. The resulting solution was stirredfor 17 hours at room temperature. A white solid formed which wascollected by filtration and dried to give the title compound (0.43 g),m.p. 250-252° C.

EXAMPLE 4(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,3-form (see Preparations 1, 3 and 6) (0.5 g) was dissolved in water (4ml) and methanol (4 ml) was added. The resulting solution was stirredfor 17 hours at room temperature. A white solid formed which wascollected by filtration and dried to give the title compound (0.43 g),m.p. 249-251° C.

EXAMPLE 5(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

To a solution of the compound of Preparation 12 (2.50 g, 3.20 mmol) in1,4-dioxane (20 ml) was added a solution of 1,4-dioxane (20 ml)saturated with HCl gas. After 30 minutes, the initially clear solutiondeposited an oil which was stirred for 24 hours at room temperature.Water (20 ml) was added to give a clear solution which was stirred atroom temperature for 60 hours. Evaporation of the resulting solutionunder reduced pressure gave an oil which was dissolved in water andbasified with aqueous sodium hydroxide solution until pH 4 was obtained.The solvent was removed by evaporation under reduced pressure andgranulation of the resultant material with methanol provided anoff-white solid which was collected by filtration and reslurried inwater (4 ml) overnight. The solids were filtered off and dried to yieldthe title compound (0.97 g), m.p. 225-230° C. IR and PXRD analysisconfirmed the product to be the required α-form.

EXAMPLE 6(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

To a solution of the compound of Preparation 13 (1.78 g) in 1,4-dioxane(18 ml) was added aqueous 4M hydrochloric acid (18 ml). The clear yellowsolution was allowed to stir at room temperature for 60 hours followedby an additional 18 hours at 35° C. Removal of the solvent under reducedpressure gave 5.42 g of material, 4.22 g of which was dissolved in water(10 ml), the solution basified with aqueous sodium hydroxide solution topH 4.0, seeded with the compound of Example 1 and stirred at roomtemperature for 18 hours. The resulting clear solution was concentratedto about 10 ml in volume under reduced pressure, diluted with methanol(15 ml) and granulated for 48 hours. The solids were collected byfiltration and dried to provide the title compound (1.25 g), m.p.232-235° C.

IR and PXRD analysis confirmed the product to be the required α-form.

EXAMPLE 7(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form

To a cooled (10° C.) solution of tert-butyl(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-(tert-butoxycarbonyl)propyl]-1-cyclopentylcarbonyl)-O⁴-tert-butyltyrosinate(13.3 g, 15.0 mmol) in ethyl acetate (27 ml) was added a 5.1M solutionof hydrogen chloride in ethyl acetate (70 ml) (357 mmol of HCl). After30 minutes the initially clear solution deposited a tar. The mixture wasstirred at room temperature for 18 hours. The clear solution wasdecanted off from the tar and the tar triturated with ethyl acetate (75ml) to give a sticky solid. The decantation and trituration wererepeated 5 times to give a hygroscopic solid which was dissolved inwater (12 ml). The resulting aqueous solution was washed twice withethyl acetate, basified with aqueous sodium hydroxide solution to pH4.0, seeded with the compound of Example 1 and stirred at 45-50° C. for42 hours. The off-white solids were collected by filtration, washed withwater and acetone and dried to give the title compound (1.95 g), m.p.237-238° C.

IR and PXRD analysis confirmed the product to be the required α-form.

The following Preparations illustrate the preparation of certainintermediate compounds used in synthesising the α-form:

PREPARATION 1(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,β-form

A solution of(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-carboxypropyl]-1-cyclopentylcarbonyl)tyrosine(see Preparation 9) (371 g) in a 9:1 ethanol/water mixture (2.225 l) washydrogenated at 414 kPa (60 psi) and room temperature over a 10%palladium-on-carbon catalyst (37.0 g) for 4 hours. The catalyst wasfiltered off and the filtrate evaporated to leave the crude product as afoam. This material was stirred with acetone (3.13 l) for 24 hours togive the title compound as a white amorphous solid (283 g). Found:C,52.97; H,7.02; N,8.97. C₂₆H₄₀N₄O₉S requires: C,53.41; H,6.90; N,9.58%.

PREPARATION 2(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinehydrate (the δ-form)

A solution of(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-carboxypropyl]-1-cyclopentylcarbonyl)tyrosine(see Preparation 9) (351 g) in ethyl acetate (1300 ml) was added towater (385 ml) and the two phase mixture hydrogenated at 414 kPa (60psi) and room temperature over a 5% palladium-on-carbon catalyst (35 g)for 20 hours. The catalyst was filtered off, the aqueous phase separatedand concentrated to low volume under reduced pressure. The viscoussolution was poured into methanol (2.85 l) and stirred at roomtemperature for 18 hours during which time there was a slowprecipitation of a solid. The solid was granulated at 5-10° C. for 2hours, filtered, washed with methanol and dried to give the titlecompound as a white solid (178.1 g), m.p. 168-171° C. Found: C,51.37;H,7.47; N,9.06. C₂₆H₄₀N₄O₉S.χH₂O (where χ=1) requires: C,51.81; H,7.02;N,9.30%.

Water content=3.6% by weight as determined by Karl Fischer analysis (χ=1requires 3.0% by weight).

PREPARATION 3(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,β-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinehydrate (the δ-form, see Preparation 2) (20.0 g) was dissolved in water(250 ml) at room temperature and the clear solution frozen using a solidcarbon dioxide/acetone bath. The solid mass was freeze dried to yieldthe title compound as a white solid (19.0 g). This material decomposedslowly over the temperature range 155-170° C.

PREPARATION 4(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,γ-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinehydrate (the δ-form, see Preparation 2) (1.0 g) was stirred with eithern-propanol or acetonitrile (10 ml) for 24 hours at room temperature. Ineach case the white solid obtained was collected by filtration and driedto provide the title compound, m.p. 172-176° C.

PREPARATION 5(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,γ-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosinehydrate (the δ-form, see Preparation 2) (847.0 g) was dissolved in water(762 ml) and the solution diluted with acetone (1.0 l). This solutionwas added slowly to vigorously stirred acetone (18.05 l) at roomtemperature and a white solid precipitated. The mixture was stirred atroom temperature for 18 hours, the solid was collected by filtration,washed with acetone and dried to give the title compound as a whitesolid (775 g), m.p. 179-181° C. Found: C,53.42; H,6.88; N,9.37; S,5.49.C₂₆H₄₀N₄O₉S requires: C,53.41; H,6.90; N,9.58; S,5.48%.

PREPARATION 6(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,β-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,α-form (see Examples 1 to 4) (4.0 g) was added to water (200 ml) and themixture stirred at 90-95° C. for 30 minutes. Insoluble material wasfiltered off, the filtrate diluted with further water (50 ml) and cooledto room temperature. After filtration to remove a slight haze, the clearfiltrate was frozen using a solid carbon dioxide/acetone bath. The solidmass obtained was freeze dried to yield the title compound as a whitesolid (3.0 g). This material decomposed slowly over the temperaturerange 155-165° C.

PREPARATION 7(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,γ-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine,β-form (see Preparations 1, 3 and 6) (0.3 g) was slurried inacetonitrile (15 ml) and stirred for 5 days. The resulting white solidwas collected by filtration and dried under reduced pressure to providethe title compound (0.26 g).

PREPARATION 8(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl)-1-cyclopentylcarbonyl)tyrosine,γ-form

(S,S,S)-N-(1-[2-Carboxy-3-(N²-mesyllysylamino)propyl)-1-cyclopentylcarbonyl)tyrosine,β-form (see Preparations 1, 3 and 6) (0.3 g) was slurried in n-propanol(10 ml) and stirred for 5 days. The resulting white solid was collectedby filtration and dried under reduced pressure to provide the titlecompound (0.26 g), m.p. 175-180° C.

PREPARATION 9(S,S,S)-N-(1-[3-(N⁶-Benzyloxycarbonyl-N²-mesyllysylamino)-2-carboxypropyl]-1-cyclopentylcarbonyl)tyrosine

Tert-butyl(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-(tert-butoxycarbonyl)propyl]-1-cyclopentylcarbonyl)-O⁴-tert-butyltyrosinate(404 g) was dissolved in dichloromethane (810 ml). Anisole (769 g) wasadded in one portion and then trifluoroacetic acid (1.158 kg) addeddropwise over approximately 10 minutes. On completion of the addition,the reaction was stirred at 35° C. for 6 hours and then stirred at roomtemperature overnight. Water (1000 ml) was added and three layersformed. The top and bottom layers were combined, dissolved in ethylacetate (2 l) and the resulting solution washed with brine. The organicphase was mixed with brine, the pH adjusted to 3 and the layers allowedto separate. Three layers formed. The organic phases were separated,taken up in ethyl acetate and extracted with saturated aqueous sodiumbicarbonate (1.6 l) solution and brine (0.5 l). The combined aqueouslayers were washed with ethyl acetate, then acidified and extracted withethyl acetate to give an ethyl acetate solution (1.54 l) of the titlecompound. This solution was either used directly (e.g. see Example 1) orthe solvent removed to provide the title compound.

PREPARATION 10 (S)-N⁶-Benzyloxycarbonyl-N²-mesyllysine

(S)-N⁶-Benzyloxycarbonyllysine (1.5 kg) was slurried in methylenechloride (7.5 l) and chlorotrimethylsilane (1.36 l) added over 10minutes. The mixture was heated under reflux for 30 minutes to give asolution which was cooled to 3° C. before simultaneously addingdiisopropylethylamine (1.87 l) and methanesulphonyl chloride (435 ml) atsuch a rate as to keep the temperature below 25° C. The reaction wasstirred for a further 2.5 hours then poured into 2 M aqueoushydrochloric acid solution. The layers were separated and the methylenechloride phase was washed with 2 M aqueous hydrochloric acid solutionfollowed by water. The solvent was removed under reduced pressure andreplaced with n-butyl acetate. The solution was cooled and the resultingcrystalline material was collected by filtration, washed with n-butylacetate and dried under reduced pressure to provide the title compound(1.63 kg), m.p. 83.5-84° C. [α]_(D) ²⁵−13.4° (c=1, methanol). Found:C,50.23; H,6.40; N,7.76.

C₁₅H₂₂N₂O₆S requires: C,50.27; H,6.19; N,7.82%. ¹H-NMR (300 MHz,d₆-DMSO): δ=1.23-1.78(6H,m), 2.85(3H,s), 2.98(2H,q), 3.80(1H,dt),5.00(2H,s), 7.25(1H,t), 7.30-7.43(5H,m), 7.51(1H,d) ppm.

PREPARATION 11 Tert-butyl(S,S,S)-N-(1-[2-tert-butoxycarbonyl-3-(N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)-O⁴-tert-butyltyrosinate

To a solution of tert-butyl(S,S,S)-N-(1-[3-(N⁶-benzyloxycarbonyl-N²-mesyllysylamino)-2-(tert-butoxycarbonyl)propyl]-1-cyclopentylcarbonyl)-O⁴-tert-butyltyrosinate(48.64 g, 54.8 mmmol) in industrial methylated spirits (1.0 L) was added5% palladium-on-carbon (5 g) (water wet) and the mixture washydrogenated at 345-414 kPa (50-60 psi) and at room temperature for 19hours. After removal of the catalyst by filtration, the resultingsolution was concentrated under reduced pressure to provide the titlecompound as a colourless oil (46.56 g) which contained ethanol.

¹H-NMR (300 MHz, CDCl₃): δ=1.27(9H,s), 1.41(9H,s), 1.44(9H,s),1.45-1.62(14H, broad m), 1.8-2.05(4H, broad m), 2.21(2H,m), 2.72(2H,t),2.79(3H, broad), 2.96(3H,s), 3.1(2H,m), 3.59(1H,m), 3.96(1H,t),4.73(1H,m), 6.43(1H,dt), 6.89(2H,dt), 7.09(2H,dt), 7.51(1H,dt) ppm.

PREPARATION 12 Tert-butyl(S,S,S)-N-(1-[2-tert-butoxycarbonyl-3-(N⁶-formyl-N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)-O⁴-tert-butyltyrosinate

A cooled (0° C.) solution of formic acetic anhydride in acetic acid(made by combining 45.3 ml of acetic anhydride with 22.8 ml of formicacid, heating the resulting solution to 50-60° C. for 15 minutes, thencooling to 0° C.) was added to a solution of the compound of Preparation11 (27.3 g, 36.3 mmol) in formic acid (33.7 ml) at 0° C. over 10minutes. The solution was allowed to warm to and stirred at roomtemperature for 45 minutes and then quenched onto ice. The resultingmixture was neutralised with aqueous sodium hydroxide solution andextracted with dichloromethane (x 2). The combined organic layers werewashed twice with brine and evaporated under reduced pressure to providethe title compound as a yellow foam (28.0 g).

¹H-NMR (300 MHz, CDCl₃): δ=1.26(9H,s), 1.41(18H,s), 1.45-2.03(16H,broad), 2.23(2H, broad m), 2.97(3H,s), 3.08(2H,m), 3.28(2H,m),3.51(1H,m), 3.98(1H, broad m), 4.73(1H,q), 5.57(1H, broad dt), 5.91(1H,broad), 6.32(1H,dt), 6.90(2H,dt), 7.08(2H,dt), 7.29(1H,broad),8.17(1H,s) ppm.

PREPARATION 13(S,S,S)-N-(1-[2-Carboxy-3-(N⁶-formyl-N²-mesyllysylamino)propyl]-1-cyclopentylcarbonyl)tyrosine

To a cooled (0° C.) solution of the compound of Preparation 12 (2.71 g,3.46 mmol) in dichloromethane (4.8 ml) was added trifluoroacetic acid(4.8 ml). The reaction was allowed to warm to room temperature andstirred for 24 hours. The mixture was then concentrated under reducedpressure to provide the title compound as a solid (2.4 g), m.p. 56-60°C.

¹H-NMR (300 MHz, d₆-DMSO): δ=1.2-1.6(14H, broad m), 1.71-1.86(3H,m),1.86-1.99(1H,m), 2.28-2.41(1H,m), 2.78(3H,s), 2.8-3.09(4H,m),3.12-3.25(2H,m), 3.7(1H,m), 4.35(1H,m), 6.6(2H,dt), 6.98(2H,dt),7.25(1H,dt), 7.50(1H,dt), 7.91(2H,m), 7.97(1H,s) ppm.

Characterisation of the α-, β-, γ- and δ-forms by IR, PXRD and DSCAnalysis and by Melting Point Determination

a) Infra-red Spectroscopy (IR)

The infra-red spectra of the different forms were determined as nujolmulls using a Nicolet 800 FT-IR spectrometer. For each form, the wavenumbers (v [cm⁻¹]) of the absorption bands are listed in Table 1.

TABLE 1 α-form β-form γ-form δ-form 3667* 3407* 3425* 3386* 3384 33773380 3223 3240 3287 3153 3137 3098 1708 1709 1699 1673* 1652* 1665* 16381639 1637 1626 1615 1619 1594 1595 1594 1596 1568 1556 1533 1527 15161516 1518 1516 1494* 1457 1458 1457 1458 (nujol) (nujol) (nujol) (nujol)1443 1448 1419 1396 1390 1377 1378 1377 1378 (nujol) (nujol) (nujol)(nujol) 1356 1344 1344 1334 1338 1321 1317 1313 1304 1300 1270 1267 12541241 1245 1249 1228 1229 1210 1195 1198 1172 1178 1174 1164 1162 1151*1144 1143 1141 1137 1118 1111 1109 1106 1108 1093 1098 1091 1074 10751064 1045 1046 1045 1031 1033 1019 1012 1019 1003 1001  981  980  985 972  965  962  962  945  941  932  911  907  909  897*  889  889  879 877  862  849  841  830  822  818  815  800  808  806  807  780  778 762  763  753  744  737  732  721  721  729  721  665  655  658  655*indicates those bands which are considered to be the most significantin terms of differentiating between the various forms.

 Representative infra-red spectra for the various forms are shown inFIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B.

(b) Powder X-ray Diffraction (PXRD)

The powder X-ray diffraction patterns of the various forms were obtainedusing a Siemens D500 diffractometer that was operated at 40 kV/30 mA andusing copper radiation filtered with a graphite monochromator (λ=0.15405nm) and a scintillation counter detector. For each form, beam intensityas a function of the angle 2θ was recorded over the range 20° to 45° 2θusing a step scan mode counting for six seconds at step intervals of0.03° 2θ. For each form, the main peaks (degrees 2θ) seen in the patternare listed in Table 2.

TABLE 2 α-form γ-form δ-form (sharp (sharp (sharp peaks) (β-form peaks)peaks)  7.5  8.9  9.9 Broad  9.0, 9.6 peaks with 10.6 10.5, 10.8 11.6centres at 11.6 11 and 20 12.7 12.3 13.3 14.6 14.5 15.6 16.2 17.2, 17.517.9 17.2, 17.6, 17.9 18.0 18.8 18.9 20.2 20.2 20.4 21.8 21.5 22.1 22.423.3 23.0, 23.1 24.7 27.1, 27.8 28.9

 Representative powder X-ray diffraction patterns for the various formsare shown in FIGS. 5 to 8.

(c) Differential Scanning Calorimetry (DSC)

Samples (about 5 mg) of the various forms were analysed using aPerkin-Elmer 7 Series thermal analyser at a scanning rate of 20° C. perminute. The results obtained for the various forms are summarised inTable 3.

TABLE 3 Form Summary of DSC analysis α-form Sharp endotherm in the range248-259° C. Decomposition above 260° C. β-form Broad endotherm in therange 60-130° C. Weak endotherm at about 147° C. Decomposition above200° C. γ-form Sharp endotherm in the range 176-186° C. Sharp exothermat about 207° C. Weak endotherm at about 213° C. Decomposition above250° C. δ-form Sharp endotherms at about 162 and at about 166-168° C.Decomposition above 200° C.

 Representative DSC thermograms for the various forms are shown in FIGS.9 to 12.

(d) Melting Point

The melting points of the various forms were determined by hot stagemicroscopy using a Mettler FP5/FP52 apparatus at a heating rate of 2° C.per minute. The typical ranges within which the various forms melt areset out in Table 4.

TABLE 4 Sharp melting points Form in the range (° C.) α-form 242-252γ-form 170-185 δ-form 165-175

Comparative Studies

The α- and β-forms were compared using processing and hygroscopicitystudies.

(a) Processing Study

An instrumented tablet machine (Manesty Machines Limited, Model F3) wassatisfactorily calibrated for force and upper punch displacement.

When calibrated, a placebo Avicel (trade mark)/DCP (dibasic calciumphosphate) blend was processed on the machine using 13 mm flat facedpunches to measure the reproducibility of the technique. Using analiquot of the blend, the machine was adjusted appropriately to achievethe target compression weight (400 mg) and sufficient hardness. Twentyunit aliquots were then separately weighed and loaded into the shoe ofthe machine. The machine was operated under power until the blend in theshoe had been exhausted and no further tablets were produced. FIG. 13shows a plot of upper punch force as a function of the number of tabletsfor three Avicel/DCP placebo blends, each of twenty units, and Table 5shows the mean weight and hardness of the ten heaviest tablets (assumedto be the first ten produced). It can be seen from the data presented inFIG. 13 that the overall process, for this blend, was very reproducible.The decrease in upper punch force that occurred at the end of the runcan be correlated with the reduction in the amount of blend in the shoeand consequential poor filling of the die.

TABLE 5 Table showing the mean weight and hardness of tablets producedusing an Avicel/DCP placebo blend. Mean Mean weight Standard hardnessStandard Run (mg) Deviation (kPa) Deviation 1 394.3 7.82 16.0 1.68 2389.6 9.20 14.6 2.04 3 393.3 6.93 15.1 1.22

Following the experiment to determine the reproducibility of thetechnique, blends containing the α-form or the β-form were separatelyprepared according to the following formulation: α- or β-form (100 mg),pregelatinsed starch (40 mg), dibasic calcium phosphate (anhydrousgrade) (256 mg) and magnesium stearate (2 mg). A blend/screen/blendprocess was used to manufacture 20 g of the blend prior to slugging onthe machine. The loading was 100 mg as previous experience had indicatedthat the higher the loading, the more processing difficulties that wereencountered. The machine was adjusted for the blend and then 50 tabletswere produced from the particular blend in one continuous batch.

Optimisation of the machine was more difficult with the β-form blend dueto its poor flow properties. Despite careful manipulation of the processvariables, it was not possible to maintain the upper punch forceconstant between both blends and consequently the β-form blend wascompressed to a greater hardness.

The upper punch data are shown for both blends in FIG. 14. The largevariability in upper punch force (and tablet weight) for the β-formblend was associated with the non-uniform filling of the die for thisformulation. The data presented in Table 6 confirms that processing ofthe β-form formulation was much more difficult and was subject to muchgreater variability than if the α-form formulation was used.

TABLE 6 Table showing the variability in processing parameters forblends containing the α- and β-forms. Mean upper Mean punch tablet Meanforce Standard weight Standard hardness Standard Sample (kN) Deviation(mg) Deviation (kP) Deviation α-form 18.0 1.85 398 17.6  5.0 1.15 blendβ-form 23.2 7.07 446 48.7 18.5 4.69 blend

The measured ejection force for the last ten tablets of each blend isshown in FIG. 15. The tablets formed from the β-form required muchgreater force to remove them from the die. This effect manifested itselfin the tablets being “flipped” from the die by the shoe.

The data obtained shows the poor processing properties of the β-form ascompared to the α-form. The β-form has a low bulk density (fluffdensity=0.09 g ml⁻¹, compared with 0.36 g ml⁻¹ for the α-form) and poorflow properties and when blends containing it are tabletted, a largevariability in tablet weight results and a high ejection force isrequired. In all these respects, the α-form has been shown to exhibitsuperior properties making it particularly suitable for pharmaceuticalformulation.

(b) Hygroscopicity Study

(i) The hygroscopicity of the α- and β-forms was assessed by gravimetricanalysis as follows.

Samples of the α- and β-forms were separately placed in Kilner (trademark) jars under the following conditions: 40° C.; 40° C. and 75%RH(relative humidity); and 40° C. and 95%RH. Water uptake of each samplewas assessed gravimetrically, in triplicate, after selected timeintervals.

Samples of the β-form stored at 40° C./75%RH or 40° C./95%RH for 1 dayunderwent a morphological change. Samples of the β-form stored at 40°C./95%RH for 1 day underwent a small weight loss (presumably after aweight increase due to water absorption followed by the morphologicalchange and then moisture loss), whereas samples stored at 40° C./75%RHgained, on average, 6% of their original weight.

FIG. 16 shows the results obtained from the gravimetric analysis. Theα-form was not found to be hygroscopic. However the β-form was found tobe very hygroscopic at 40° C./75%RH.

(ii) Moisture microbalance experiments on the α- and β-forms confirmedthat the α-form was not hygroscopic whereas the β-form was veryhygroscopic.

Samples of the α- and β-forms were separately placed in the apparatus at40° C. and allowed to equilibrate with the surroundings prior to theparticular sample being exposed to increasing relative humidities, withequilibration periods between each increase in humidity.

The results are shown in FIG. 17. These indicate that as much as 8% byweight of water (cf. original weight) was taken up by the β-form duringthe experiment.

The morphological change that the β-form underwent at high humiditieswas further studied and a transformation from a very low bulk densitypowder to a dense glassy solid was observed.

We claim:
 1. A crystalline, α-polymorphic form of a compound of theformula:

characterised by an infra-red spectrum as a mull in nujol which showsabsorption bands at ν=3407, 3386, 3223, 3153, 1699, 1652, 1626, 1594,1516, 1457 (nujol), 1377 (nujol), 1344, 1334, 1317, 1267, 1241, 1228,1210, 1164, 1151, 1137, 1118, 1109, 1093, 1074, 1045, 1019, 1003, 981,965, 911, 897, 862, 818, 800, 778, 762, 721 and 655 cm⁻¹.
 2. A compoundas claimed in claim 1 which is further characterised by a powder X-raydiffraction pattern obtained using copper radiation filtered with agraphite monochromator (λ=0.15405 nm) which shows main peaks at 7.5,8.9, 9.9, 11.6, 15.6, 17.2, 17.5, 18.0, 20.2, 22.1 and 23.3 degrees 2θ.3. A γ-polymorphic form of a compound of the formula (I) as defined inclaim 1 characterised by an infra-red spectrum as a mull in nujol whichshows absorption bands at ν=3377, 3240, 1665, 1639, 1594, 1527, 1518,1494, 1457 (nujol), 1443, 1377 (nujol), 1344, 1321, 1304, 1254, 1195,1178, 1162, 1143, 1111, 1098, 1046, 1031, 1012, 972, 962, 945, 932, 907,879, 849, 815, 806, 780, 753, 729 and 658 cm⁻¹.
 4. A compound as claimedin claim 3 which is further characterised by a powder X-ray diffractionpattern obtained using copper radiation filtered with a graphitemonochromator (λ=0.15405 nm) which shows main peaks at 9.0, 9.6, 10.6,11.6, 12.7, 13.3, 14.6, 16.2, 17.9, 18.8, 20.2 and 21.8 degrees 2θ.
 5. Ahydrated δ-form of a compound of the formula (I) as defined in claim 1characterised by a water content of from 1 to 7% by weight, asdetermined by Karl Fischer analysis, and an infra-red spectrum as a mullin nujol which shows absorption bands at ν=3667, 3425, 3380, 3287, 3137,3098, 1709, 1673, 1637, 1619, 1596, 1568, 1556, 1516, 1458 (nujol),1448, 1419, 1390, 1378 (nujol), 1356, 1338, 1300, 1270, 1249, 1229,1198, 1174, 1141, 1108, 1091, 1075, 1064, 1045, 1033, 1019, 1001, 985,962, 941, 909, 889, 877, 841, 822, 807, 763, 744, 732, 721 and 655 cm⁻¹.6. A compound as claimed in claim 5 which is further characterised by apowder X-ray diffraction pattern obtained using copper radiationfiltered with a graphite monochromator (λ=0.15405 nm) which shows mainpeaks at 10.5, 10.8, 12.3, 14.5, 17.2, 17.6, 17.9, 18.9, 20.4, 21.5,22.4, 23.0, 23.1, 24.7, 27.1, 27.8 and 28.9 degrees 2θ.
 7. A compound asclaimed in claim 6 which has a water content of from 2 to 4% by weight,as determined by Karl Fischer analysis.
 8. A pharmaceutical compositioncomprising the α-polymorphic form of a compound of the formula (I) asclaimed in claim 1 or 2, the γ-polymorphic form of a compound of theformula (I) as claimed in claim 3 or 4, or the hydrated δ-form of acompound of the formula (I) as claimed in claim 5, 6 or 7, together witha pharmaceutically acceptable diluent or carrier.
 9. A composition asclaimed in claim 8 wherein the α-polymorphic form of a compound of theformula (I) is present.
 10. A method of treatment of an animal to treata disease which is dependent on the inhibition of angiotensin convertingenzyme and/or zinc dependent neutral endopeptidase E.C. 3.4.24.11hypertension, congestive heart failure, renal insufficiency whichcomprises administering to said animal a said enzyme and/or saidendopeptidase inhibitory amount of the α-polymorphic form of a compoundof the formula (I) as claimed in claim 1 or 2, the γ-polymorphic form ofa compound of the formula (I) as claimed in claim 3 or 4, or thehydrated δ-form of a compound of the formula (I) as claimed in claim 5,6 or
 7. 11. A method as claimed in claim 10 wherein the α-polymorphicform of a compound of the formula (I) or a composition thereof is used.12. A sodium, potassium, ammonium or (C₁-C₄ alkyl)ammonium salt of acompound of the formula:


13. A sodium salt of a compound of the formula (II) as claimed in claim12.
 14. A compound of the formula:

wherein P¹, P², P³ and P⁴, which may be the same or different, are allprotecting groups that are capable of removal, to provide a compound ofthe formula (I) as defined in claim 1, with the proviso that P¹ is notbenzyloxycarbonyl when P², P³ and P⁴ are each t-butyl.
 15. A compound asclaimed in claim 14 wherein P¹ is formyl or benzyloxycarbonyl.
 16. Acompound as claimed in claim 14, wherein P², P³ and P⁴ are each t-butyl.17. A compound of the formula:

wherein P⁵ is a protecting group that is capable of removal, to providea compound of the formula (I) as defined in claim 1, with the provisothat P⁵ is not benzyloxycarbonyl.
 18. A compound as claimed in claim 17wherein P⁵ is formyl.